Phospholipid Degradation Products Research Articles

Potential Phosphorus Uptake Mechanisms in the Deep Sedimentary Biosphere

Our understanding of phosphorus (P) dynamics in the deep subseafloor environment remains limited. Here we investigate potential microbial P uptake mechanisms in oligotrophic marine sediments beneath the North Atlantic Gyre and their effects on the relative distribution of organic P compounds as a function of burial depth and changing redox conditions. We use metagenomic analyses to determine the presence of microbial functional genes pertaining to P uptake and metabolism, and solution 31P nuclear magnetic resonance spectroscopy (31P NMR) to characterize and quantify P substrates. Phosphorus compounds or compound classes identified with 31P NMR include inorganic P compounds (orthophosphate, pyrophosphate, polyphosphate), phosphonates, orthophosphate monoesters (including inositol hexakisphosphate stereoisomers) and orthophosphate diesters (including DNA and phospholipid degradation products). Some of the genes identified include genes related to phosphate transport, phosphonate and polyphosphate metabolism, as well as phosphite uptake. Our findings suggest that the deep sedimentary biosphere may have adapted to take advantage of a wide array of P substrates and could play a role in the gradual breakdown of inositol and sugar phosphates, as well as reduced P compounds and polyphosphates.

Analysis of verteporfin liposomal formulations for phospholipids and phospholipid degradation products by liquid chromatography-mass spectrometry (LC-MS)

Lipid composition and lipid degradation are critical to the stability of liposomal formulations which can impact the safety and efficacy of the drug. Herein we developed and validated an ultrahigh performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) method for determining phospholipid composition and phospholipid degradation products in a verteporfin liposomal formulation (Visudyne). The high mass accuracy (<5 ppm) of the QTOF method coupled with database searching (SimLipid) and comparison with known standards accurately identified and quantified the phospholipid compositions and lipid degradation products. The analysis of Visudyne indicated that more than 50% (w/w) of the total phospholipids are composed of phosphatidylcholine (PC) 14:0–14:0 and major phosphatidylglycerol (PG) species found are PG 16:0–18:2, PG 16:0–18:1, PG 18:0–18:2, and PG 18:0–18:1. The LC-MS method developed is capable of separating structural isomers such as PG 18:1–18:1 versus PG 18:0–18:2 and the separation of PG stereoisomers, such as PG 18:1–18:1 cis and PG 18:1–18:1 trans. The major lipid degradation products in Visudyne includes lysophosphatidylcholine and a few saturated and unsaturated lysophosphatidylglycerols, and free fatty acids (FFA). Each degradation product is less than 1% of the total phospholipids (w/w). In addition, the lipid profiles of naturally sourced egg PG from six different vendors were compared with the PG composition in Visudyne. Differences in lipid composition in egg PGs from different vendors were observed and the PG composition in Visudyne is matched with the lipid profile of the some of the egg PGs from different vendors. Drug developers can utilize this method to assess raw materials and lipid-based drug product quality and regulatory scientists can monitor the quality of the drug available in the market using this validated method.

Quantification of phospholipid degradation products in liposomal pharmaceutical formulations by ultra performance liquid chromatography-mass spectrometry (UPLC-MS).

Identification and quantification of excipient related degradation products in the liposomal formulation is important, as they may impact the safety and efficacy of the drug. Phospholipids are one of the major excipients in liposome drugs composing the lipid bilayer, and they are vulnerable to oxidation and hydrolysis reactions. Since phospholipids with saturated fatty acid chain were preferred in most of liposome drug products, the major degradation pathway of phospholipids in liposome formulations are limited to hydrolysis of phospholipids into free fatty acids and lysophospholipids. These hydrolyzed degradation products may form during manufacturing and/or long-term storage of liposomal formulations. Herein, we report development and application of accurate and sensitive methods that can be utilized for the quantitation of saturated free fatty acids (FFA 18:0 and FFA 16:0), lysophosphocholines (LPC 18:0 and LPC 16:0), and lysophosphoglycerol (LPG 18:0) in liposomal formulations. The free fatty acids were separated using a C8 column whereas the LPCs and LPGs were separated using a C18 stationary phase upon direct injection without the need of lipid extraction process. Each analyte was quantified by Q-TOF mass spectrometry. This method was validated according to USP compendial procedures and has been applied to the analysis of four commercial liposomal pharmaceutical formulations. The limit of quantitation (LOQs) of FFA 16:0, FFA 18:0, LPC 16:0, LPC 18:0 and LPG 18:0 are 5ng/mL, 5ng/mL, 6.5ng/mL, 7.0ng/mL, 10ng/mL respectively. Compared to CAD (Charge Aerosol Detector) and ELSD (Evaporative Light Scattering Detector) detection methods in ppm levels, this ultra-performance liquid chromatography (UPLC)-Mass Spectroscopy (MS) method displays precise determination of lysophospholipids in the liposomal formulations with higher accuracy and sensitivity.

Modifications of Membrane Phospholipids in Response to Extended Aging from Pork Loins

ObjectivesIt is well established that fresh meat shelf-life deteriorates during aging process. We hypothesize that part of the shelf-life reduction is due to membrane phospholipid deterioration through phospholipase activity and/or phospholipid oxidation during aging. Therefore, the objective of this study was to characterize the modifications/deterioration of phospholipid classes/species in pork loins from 3 different aging periods.Materials and MethodsLoins from 20 carcasses were collected at a commercial harvest facility in the Midwest 1 d postmortem from carcasses of Duroc sired crossbred pigs. Four chops from each carcass containing only the longissimus muscle were vacuum packaged and aged at 4°C for 1, 8, and 21 d. A sensitive approach based on electrospray ionization tandem mass spectrometry was used to comprehensively analyze phospholipid composition using the lipid extract from each sample at each aging period (n = 60). Unsaturation index (UI; measurement of the number of double bonds) for each phospholipid species class was also calculated to quantify fatty acyl chain unsaturation for each sample in each aging period.ResultsTotal phospholipid quantity in pork loins was not different between 1d and 8d aged chops but decreased significantly from 8d to 21d of aging (806.6 vs. 297.5 nmol phospholipid/mg lipid; P &lt; 0.01). On the other hand, the mol% data (distribution of each phospholipid species in relative % of total phospholipid) revealed that phosphatidylinositol (PI) and phosphatidylserine (PS) increased in mol% from 1d to 21d of aging in pork loins (P &lt; 0.01). This increase was mainly due to the increase of PI 38:4 (primarily 18:0/20:4) and PS 36:2 (primarily 18:0/18:2) between 1d and 21d samples (P &lt; 0.01). The results showed that phospholipid degradation products like lysophosphatidylcholine (LPC) mol% rose quickly after short term aging (8d) but remained constant through the rest of the 21d aging period (P &lt; 0.01). Conversely, lysophosphatidylethanolamine (LPE) was unaltered between 1d and 8d of aging but decreased between 8d and 21d aged pork loins (P &lt; 0.01). The mol% of phosphatidic acid (PA) also increased between 1d and 21d aged pork loins (P &lt; 0.05). Extended aging did not alter the mol% of total phosphatidylcholine (PC), ether-linked PC (ePC), sphingomyelin (SM), phosphatidylethanolamine (PE) or ether-linked PE (ePE; P &gt; 0.05). Surprisingly, UI revealed the exact opposite trend as the mol% data. The UI of PI and PS decreased (P &lt; 0.01) from 1d to 21 d of aging in pork loins due to the disappearance of many minor PI and PS species with very long chain fatty acids and multiple double bonds such as PI 42:10 and PS 44:10. There was also a slight increase of PC UI after 8 d of aging in pork loins (P &lt; 0.01). The UI for LPC, ePC, SM, LPE, PE, ePE and total phospholipid were not altered in any of the aging periods (P &gt; 0.05).ConclusionThese results confirmed our hypothesis that phospholipids undergo extensive degradation during aging. The data also indicated that the majority of phospholipids in pork loins may maintain integrity over short period aging (1–8d). Among the phospholipid classes, PI and PS were slightly more resistant to deterioration compared with the others due to their ability to modify fatty acyl chain saturation. Additional investigations are necessary to define the role of phospholipid modifications in fresh pork shelf-life and flavor.

Modifications of Membrane Phospholipids in Response to Extended Aging from Pork Loins

ObjectivesIt is well established that fresh meat shelf-life deteriorates during aging process. We hypothesize that part of the shelf-life reduction is due to membrane phospholipid deterioration through phospholipase activity and/or phospholipid oxidation during aging. Therefore, the objective of this study was to characterize the modifications/deterioration of phospholipid classes/species in pork loins from 3 different aging periods.Materials and MethodsLoins from 20 carcasses were collected at a commercial harvest facility in the Midwest 1 d postmortem from carcasses of Duroc sired crossbred pigs. Four chops from each carcass containing only the longissimus muscle were vacuum packaged and aged at 4°C for 1, 8, and 21 d. A sensitive approach based on electrospray ionization tandem mass spectrometry was used to comprehensively analyze phospholipid composition using the lipid extract from each sample at each aging period (n = 60). Unsaturation index (UI; measurement of the number of double bonds) for each phospholipid species class was also calculated to quantify fatty acyl chain unsaturation for each sample in each aging period.ResultsTotal phospholipid quantity in pork loins was not different between 1d and 8d aged chops but decreased significantly from 8d to 21d of aging (806.6 vs. 297.5 nmol phospholipid/mg lipid; P < 0.01). On the other hand, the mol% data (distribution of each phospholipid species in relative % of total phospholipid) revealed that phosphatidylinositol (PI) and phosphatidylserine (PS) increased in mol% from 1d to 21d of aging in pork loins (P < 0.01). This increase was mainly due to the increase of PI 38:4 (primarily 18:0/20:4) and PS 36:2 (primarily 18:0/18:2) between 1d and 21d samples (P < 0.01). The results showed that phospholipid degradation products like lysophosphatidylcholine (LPC) mol% rose quickly after short term aging (8d) but remained constant through the rest of the 21d aging period (P < 0.01). Conversely, lysophosphatidylethanolamine (LPE) was unaltered between 1d and 8d of aging but decreased between 8d and 21d aged pork loins (P < 0.01). The mol% of phosphatidic acid (PA) also increased between 1d and 21d aged pork loins (P < 0.05). Extended aging did not alter the mol% of total phosphatidylcholine (PC), ether-linked PC (ePC), sphingomyelin (SM), phosphatidylethanolamine (PE) or ether-linked PE (ePE; P > 0.05). Surprisingly, UI revealed the exact opposite trend as the mol% data. The UI of PI and PS decreased (P < 0.01) from 1d to 21 d of aging in pork loins due to the disappearance of many minor PI and PS species with very long chain fatty acids and multiple double bonds such as PI 42:10 and PS 44:10. There was also a slight increase of PC UI after 8 d of aging in pork loins (P < 0.01). The UI for LPC, ePC, SM, LPE, PE, ePE and total phospholipid were not altered in any of the aging periods (P > 0.05).ConclusionThese results confirmed our hypothesis that phospholipids undergo extensive degradation during aging. The data also indicated that the majority of phospholipids in pork loins may maintain integrity over short period aging (1–8d). Among the phospholipid classes, PI and PS were slightly more resistant to deterioration compared with the others due to their ability to modify fatty acyl chain saturation. Additional investigations are necessary to define the role of phospholipid modifications in fresh pork shelf-life and flavor.

Оксидативний стрес як важливий патогенетичний механізм розвитку змін репродуктивного здоров’я у жінок з соматоформними захворюваннями та порушенням вегетативного гомеостазу

The objective: to investigate the status of phospholipids and neutral lipids of blood cells in fertile aged women with violation of autonomic homeostasis and changes in reproductive health. Patients and methods. We examined 360 women with violation of autonomic homeostasis in the form of autonomic dysfunction syndrome and somatofom dysfunction of the autonomic nervous system of hypertonic, hypotonic and cardiac type and changes in reproductive health such as: artificial abortions, infertility and uterine leiomyoma. Results. The article presents data from a study of lipid peroxidation of cell membranes in fertile aged women with violation of autonomic homeostasis and changes in reproductive health. The obtained data of decay products concentrations of phospholipids and neutral lipids of cellular membranes indicate the ravages of free radical compounds to form intermediates and final products of lipid peroxidation. The results confirm the role of oxidative stress as an important pathogenetic mechanism of changes in the reproductive health of women with somatoform disorders and violations of autonomic homeostasis. Conclusion. Changes in the concentration of phospholipids, neutral lipids and lipid peroxidation products indicate a destabilizing effect on cell membranes in women with violation of autonomic homeostasis and reproductive health changes. Increasing the number of phospholipid degradation products show a decrease in the liquid component of cell membranes, which in turn changes the nature receptor interaction, as evidenced by a study hormonal and leads to disruption of feedback mechanisms concentration of hormones in the blood. Key words: oxidative stress, lipid peroxidation, women of fertile age, violations of autonomic homeostasis, changes in reproductive health.

Expression of Cell-Surface Marker ABCB5 Causes Characteristic Modifications of Glucose, Amino Acid and Phospholipid Metabolism in the G3361 Melanoma-Initiating Cell Line.

We present a pilot study aimed at determining the effects of expression of ATP-binding cassette member B5 (ABCB5), a previously described marker for melanoma-initiating cells, on cellular metabolism. Metabolic profiles for two groups of human G3361 melanoma cells were compared, i.e. wildtype melanoma cells with intact ABCB5 expression (ABCB5-WT) and corresponding melanoma cell variants with inhibited ABCB5 expression, through shRNA-mediated gene knockdown (ABCB5-KD). A comprehensive metabolomic analysis was performed by using proton and phosphorus NMR spectroscopy of cell extracts to examine water-soluble metabolites and lipids. Parametric and non-parametric statistical analysis of absolute and relative metabolite levels yielded significant differences for compounds involved in glucose, amino acid and phospholipid (PL) metabolism. By contrast, energy metabolism was virtually unaffected by ABCB5 expression. The sum of water-soluble metabolites per total protein was 17% higher in ABCB5-WT vs. ABCB5-KD G3361 variants, but no difference was found for the sum of PLs. Enhanced abundance was particularly pronounced for lactate (+ 23%) and alanine (+ 26%), suggesting an increase in glycolysis and potentially glutaminolysis. Increases in PL degradation products, glycerophosphocholine and glycerophosphoethanolamine (+ 85 and 123%, respectively), and redistributions within the PL pool suggested enhanced membrane PL turnover as a consequence of ABCB5 expression. The possibility of glycolysis modulation by an ABCB5-dependent IL1β-mediated mechanism was supported by functional studies employing monoclonal antibody (mAb)-dependent ABCB5 protein inhibition in wildtype G3361 melanoma cells. Our metabolomic results suggest that the underlying biochemical pathways may offer targets for melanoma therapy, potentially in combination with other treatment forms.

A Comprehensive Untargeted Metabonomic Analysis of Human Steatotic Liver Tissue by RP and HILIC Chromatography Coupled to Mass Spectrometry Reveals Important Metabolic Alterations

Steatosis, or excessive accumulation of lipids in the liver, is a generally accepted previous step to the development of more severe conditions like nonalcoholic steatohepatitis, fibrosis, and cirrhosis. We aimed to characterize the metabolic profile that defines simple steatosis in human tissue and to identify potential disturbances in the hepatic metabolism that could favor the switch to progressive liver damage. A total of 46 samples, 23 from steatotic and 23 from nonsteatotic human livers, were analyzed following a holistic LC-MS-based metabonomic analysis that combines RP and HILIC chromatographic separations. Multivariate statistical data analysis satisfactorily classified samples and revealed steatosis-associated biomarkers. Increased levels of bile acids and phospholipid degradation products, and decreased levels of antioxidant species, were found in steatotic livers, indicating disturbances in lipid and bile acid homeostasis and mitochondrial dysfunction. Changes in hypoxanthine, creatinine, glutamate, glutamine, or γ-glutamyl-dipeptides concentrations, suggestive of alterations in energy metabolism and amino acid metabolism and transport, were also found. The results show that the proposed analytical strategy is suitable to achieve a comprehensive metabolic profile of steatotic human liver tissue and provide new insights into the metabolic alterations occurring in fatty liver that could contribute to its predisposition to damage evolution.

Lipid metabolism in the cockroach, Periplaneta americana, is activated by the hypertrehalosemic peptide, HTH-I

Phosphatidylcholine and phosphatidylethanolamine are the major constituents of the phospholipid pool in cockroach ( Periplaneta americana) fat body and hemolymph. Both species of phospholipid are significantly decreased 6 h after injecting hypertrehalosemic hormone I (HTH-I) into the hemocoel. Loss of phospholipid is accompanied by an accumulation of the phospholipid degradation products glycerophosphorylcholine and glycerol. HTH-I also increases phospholipase activity in the hemolymph and this is thought to be responsible for the depletion of hemolymph phospholipid. Phospholipase activity peaks approximately 2 h after injection of HTH-I and returns to normal at 6 h. In vitro, total phospholipid in the fat body is decreased by HTH-I whereas the concentration of diacylglycerol displays a corresponding increase. HTH-I elevates free fatty acid levels but has no effect on triacylglycerol. These effects of HTH-I are blocked by the phospholipase inhibitor mepacrine.

Phosphorous metabolites and steady-state energetics of transformed fibroblasts during three-dimensional growth.

Rat1-T1 and MR1 spheroids represent separate transformed phenotypes originated from the same rat fibroblasts that differ in three-dimensional (3D) growth kinetics, histological structure, and oxygenation status. In the present study, (31)P-NMR spectroscopy of perfused spheroid suspensions was used to investigate cellular energetics relative to 3D growth, development of necrosis, and cell cycle distribution. Both spheroid types were characterized by a remarkably low amount of free (inorganic) phosphate (P(i)) and a low phosphocreatine peak. The ratio of nucleoside triphosphate (NTP) to P(i) ranged between 1.5 and 2.0. Intracellular pH, NTP-to-P(i) ratio, and NTP/cell remained constant throughout spheroid growth, being unaffected by the emergence of oxygen deficiency, cell quiescence, and necrosis. However, a 50% decrease in the ratio of the lipid precursors phosphorylcholine and phosphorylethanolamine (PC/PE) was observed with increasing spheroid size and was correlated with an increased G(1)/G(0) phase cell fraction. In addition, the ratio of the phospholipid degradation products glycerophosphorylcholine and glycerophosphorylethanolamine (GPC/GPE) increased with spheroid diameter in Rat1-T1 aggregates. We conclude that changes in phospholipid metabolism, rather than alterations in energy-rich phosphates, reflect cell quiescence in spheroid cultures, because cells in the inner oxygen-deficient zones seem to adapt their energy metabolism to the environmental conditions before necrotic cell destruction.

Phospholipid synthesis by Staphylococcus aureus during (Sub)Lethal attack by mammalian 14-kilodalton group IIA phospholipase A2.

Killing of gram-positive bacteria by mammalian group IIA phospholipases A2 (PLA2) requires the catalytic activity of the enzyme. However, nearly complete degradation of the phospholipids can occur with little effect on bacterial viability, suggesting that PLA2-treated bacteria can biosynthetically replace phospholipids that are lost due to PLA2 action. In the presence of albumin, phospholipid degradation products are quantitatively sequestered extracellularly. In the absence of albumin, the bacteria retain and substantially reutilize the phospholipid breakdown products and survive an otherwise lethal dose of PLA2. PLA2-treated bacteria also continue to incorporate sodium [2-(14)C]acetate into phospholipids, suggesting that the bacteria are attempting to repair the damaged membranes by de novo synthesis of phospholipids. To determine whether PLA2 action also triggers activation of bacterial lipolytic enzymes, the effects of nisin and PLA2 on the degradation of S. aureus lipids were compared. In contrast to nisin treatment, PLA2 treatment does not stimulate endogenous phospholipase activity in S. aureus. These findings show that S. aureus responds to PLA2 attack by continued phospholipid (re)synthesis by both de novo and salvage pathways. The fate of PLA2-treated S. aureus therefore appears to depend on the relative rates of phospholipid degradation and synthesis.

Influence of degraded phosphatidylserine on binding of antiphospholipid antibodies.

Antiphospholipid antibodies (aPL) show great heterogeneity. Different phospholipids, with or without protein cofactor(s), and phospholipid binding proteins alone have been proposed as the target molecules for aPL. In order to determine the influence of phospholipid degradation products on the binding of aPL, sera from 6 patients with the antiphospholipid syndrome were studied. Fresh and aged phosphatidylserine and cardiolipin were used as coating reagents in solid-phase immunoassay procedures. Antibody reactivity was tested by enzyme-linked immunosorbent assay in the sera and in eluates from columns packed with polystyrene scrapings coated with either cardiolipin or phoshatidylserine. Three reaction patterns of affinity-purified antibodies were seen: (1) reactivity with phosphatidylserine but not with cardiolipin or degraded phosphatidylserine, (2) reactivity with cardiolipin and degraded phosphatidlyserine, and (3) reactivity with all three phospholipid antigens. Striking differences in the antiphospholipid antibody reactivity with cardiolipin, phosphatidylserine and degraded phosphatidylserine in the presence of serum proteins were observed among patients with venous thromboembolism. The analyses showed that the degradation of phosphatidylserine influences the binding of aPL in in vitro assays.

Phosphatidylcholine hydrolysis and protein kinase C activation for intracellular signaling network

Agonist-stimulated inositol phospholipid hydrolysis by phospholipase C was once thought to be the sole mechanism to produce diacylglycerol that transduce extracellular signals into intracellular events through activation of protein kinase C. It is becoming clear that agonist-induced hydrolysis, of the other membrane phospholipids, particularly phosphatidylcholine, by phospholipase A 2 and phospholipase D also takes part in cellular responses such as cell proliferation and differentiation. Possibly, the members of the protein kinase C family may be activated differently by various combinations of phospholipid degradation products, and play each distinct role in signal transduction for the control of various cellular functions.

The effects of creatine phosphate in experimental myocardial infarction

Several clinical and experimental studies have demonstrated the protective mechanisms of exogenous high-energy compounds in treating total or regional myocardial ischemia. To evaluate the effects of creatine phosphate (CP) in an experimental model, rabbits of the New Zealand line were randomly divided into two CP-treated groups (A and B) and one control group (C). In all animals the circumflex coronary artery was ligated, and the size of the infarct zone was planimetrically determined. The contents of the phospholipid degradation products also were measured. Continuous electrocardiogram monitoring revealed a statistically significant reduction in the total number of ventricular ectopic beats in groups A and B and a longer period of both ventricular tachycardia and ventricular fibrillation in the control group than in the treated groups. Our results showed a statistically significant reduction of the necrotic zone in both treated groups versus the control group. Analysis of the degradation products of phospholipid metabolism emphasized an increase of both lysophosphatidylcholine and lysophosphatidylethanolamine in the untreated group versus treated groups. Significant correlation was found between total ventricular ectopic beats and the lysophosphoglyceride contents in groups A and B. On this basis we believe that CP may prevent the ischemic consequences on the myocardium of arrhythmias resulting from electrophysiological alterations produced by an increase of lysophosphoglycerides, and should reduce the extension of the necrotic zone, probably preserving the integrity of the cell membrane.

Hepatocellular carcinoma cells resist necrosis during anoxia by preventing phospholipase-mediated calpain activation

Although hepatocellular carcinoma (HCC) cells are more resistant to anoxic injury than normal hepatocytes, the mechanisms responsible for this differential sensitivity remain obscure. Because enhanced calpain protease activity contributes to hepatocyte necrosis, we tested the hypothesis that HCC cells resist anoxia by preventing calpain activation. Cell viability in two rat HCC cell lines (N1S1 and McA-RH7777 cells) was fourfold greater compared to rat hepatocytes after 4 h of anoxia. Although calpain activity increased twofold in rat hepatocytes during anoxia, no increase in calpain activity occurred in HCC cells. Western and Northern blot analysis revealed greater or equivalent expression of calpains and calpastatin in HCC cells compared to hepatocytes. Because increases in cytosolic free Ca++ (Cai++) and phospholipid degradation products regulate calpains in vitro, we measured Cai++ and phospholipid degradation. Ca++i did not change in any cell types during 60 min of anoxia. In contrast, phospholipid degradation was fourfold greater in hepatocytes compared to HCC cells. Melittin, a phospholipase A2 activator, increased calpain activity and cell necrosis in all cell types; melittin-induced cell necrosis was ameliorated by a calpain protease inhibitor. In summary, these data demonstrate for the first time 1) calpain activation without a measureable increase in Ca++i, 2) phospholipase-mediated calpain activation in hepatocytes and HCC cells, and 3) the adaptive mechanism responsible for the resistance of HCC cells to anoxia-an inhibition of phospholipid-mediated calpain activation. Interruption of phospholipase-mediated calpain activation may be a therapeutic strategy for preventing anoxic cell injury.

A cascade of degradative hydrolase activity contributes to hepatocyte necrosis during anoxia.

Calpain proteases contribute to hepatocyte necrosis during anoxia. Our aim was to ascertain the mechanism causing calpain activation during anoxia. In rat hepatocytes, a twofold increase in calpain activity occurred despite the lack of an increase in cytosolic Ca2+ concentration ([Ca2+]i). The increase in calpain activity was not associated with an increase in calpain mRNA or a decrease in calpastatin mRNA expression. Because phospholipid degradation products generated by phospholipases can activate calpains at physiological [Ca2+]i, we determined the effect of phospholipase inhibitors and activators on calpain activity. Pretreatment of hepatocytes with fluphenazine, a phospholipase inhibitor, decreased calpain activation and improved cell survival. Melittin, a phospholipase A2 activator, increased calpain activity and potentiated cell killing. Finally, phospholipid degradation preceded the increase in calpain activity. Thus the enhanced calpain activity occurring in hepatocytes during anoxia 1) is regulated at the posttranslational level and 2) appears to be dependent on phospholipase activity. These data suggest a novel cascade for degradative hydrolase activity during hepatocyte necrosis by anoxia with phospholipase-mediated activation of calpains.

Toward a Comprehensive Model for Lipid Peroxidation in Plant Tissue Disorders

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Isolation and characterization of deteriosomes from rat liver

Deteriosomes, a new class of microvesicles, have been isolated from rat liver tissue. These microvesicles are similar to those isolated previously from plant tissue [Yao et al., Proc Natl Acad Sci USA 88:2269-2273, 1991] in that they are nonsedimentable and enriched in membrane catabolites, particularly products of phospholipid degradation. Liver deteriosomes range in size from 0.05 microns to 0.11 microns in radius. They are also much more permeable than microsomal membrane vesicles indicating that the deteriosome bilayer is perturbed. The data are consistent with the proposal that deteriosomes are formed from membranes by microvesiculation and that they represent an intermediate stage of membrane deterioration. Furthermore, liver deteriosomes were found to contain phospholipase A2 activity. This suggests that they not only serve as a means of moving destabilizing macromolecular catabolites out of membranes into the cytosol but also possess enzymatic activity. The fact that the specific activity of phospholipase A2 is higher in deteriosomes than in deteriosome-free cytosol suggests that some of the enzymatic activity traditionally assumed to be cytosolic may in fact be associated with deteriosomes.

Protein kinase C, calcium and phospholipid degradation

In most cells, calcium signals are transient, while the resulting physiological responses often persist longer. The sustained activation of protein kinase C has been postulated to be essential for maintaining such cellular responses. It is becoming clear that an elaborate network involving protein kinase C, calcium and degradation of membrane phospholipids may generate several molecules that are necessary for sustaining the activation of protein kinase C itself. Multiple members of the protein kinase C family show distinct responses to calcium and the phospholipid degradation products, suggesting their unique functions in cell signalling.

Physicochemical Characterization of a Model Intravenous Oil-in-Water Emulsion

Photon correlation spectroscopy, nuclear magnetic resonance spectroscopy, electron microscopy, and small-angle X-ray scattering were used for the structural characterization of a model oil-in-water emulsion containing particles in the submicrometer size range. Additionally, small-angle X-ray diffraction, wide-angle X-ray diffraction, and differential scanning calorimetry were applied to raw materials and to binary mixtures. The majority of emulsion droplets have the characteristic of an ideal emulsion droplet, that is, a liquid lipid core covered by an emulsifier monolayer. However, the system contains a certain excess of emulsifier. Particles with bi- and/or oligolayer structures can be deduced. Double-emulsion droplets were detected. Large unilamellar vesicles were not found; however, the existence of small unilamellar liposomes (also referred to as small unilamellar vesicles or SUVs) seems likely. The proportion of all small nonmonolayer structures was quantified by nuclear magnetic resonance spectroscopy. No mixed micellar structures were detectable. Lysophospholipids were not detected in the aqueous phase, indicating their predominant incorporation into the emulsifier layers. Water-soluble phospholipid degradation products were found in the water phase. The existence of at least several monolayers of phospholipids does not seem to be a prerequisite for a stable soybean oil-in-water emulsion, in general.